Rod dark adaptation requires the deactivation and regeneration of bleached (photoactivated) rhodopsin. Rhodopsin regeneration, i.e., the binding of 11-cis retinal by opsin, is known to depend on the recycling of retinoid within the eye tissues. However, two fundamental issues relevant to this process remain unresolved: (1) the identify of the retinoid cycle event that rate-limits regeneration in vivo, and (2) the role of regeneration in the shut-off of bleaching desensitization. The proposed project addresses these two issues through biochemical and electroretinographic (ERG) studies on mouse and toad, and in ERG studies on human subjects. A major thrust is to infer human rod recovery mechanisms based on correlations with data obtained from mouse. ERG experiments will employ a new, noninvasive, paired-flash ERG method to derive the full time course of the in vivo rod response to a test flash. Kinetic and sensitivity properties of ERG-derived human and mouse rod responses will be analyzed in relation to illumination conditions and post-bleach recovery time. Parallel biochemical experiments on mice will determine the kinetics of retinoid cycle reactions and rhodopsin regeneration. Combined ERG and biochemical results will serve to test the hypothesis (1, above) that regeneration in vivo is rate-limited by the enzymatic reduction of all-trans retinal in the rods, the delivery of 11-cis retinal to the rods, or a combination of these. The data will also resolve (2, above) whether the regeneration event shuts off bleaching desensitization or merely re-establishes maximal quantum capture efficiency. A further thrust of the project is to test the recent hypothesis that docosahexaenoic acid (DHA) regulates the activity of interphotoreceptor retinoid-binding protein (IRBP), an extracellular (interphotoreceptor matrix; IPM) protein known to support retinoid transfer between the retinal pigment epithelium (RPE) and the rods in the retinoid visual cycle. This will be addressed by testing the effect of added DHA on IRBP's retinoid-transfer activity in toad RPE-eyecups and related reconstituted preparations, and by testing for correlations between endogenous DHA level in the IPM and retinoid cycle operation in the toad and mouse eye. Results of the project will advance fundamental knowledge of dark-adaptation recovery in normally functioning rods. The ERG data will provide immediate foundation for studies of rod recovery abnormalities in human retinal disease and in animal models such as transgenic mice.